Download Enhanced T cell activation in Plasmodium falciparum malaria

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(8): 985-992, December 2012
985
Enhanced T cell activation in Plasmodium falciparum malaria-infected
human immunodeficiency virus-1 patients from Mozambique
Helena Chavale1,2, Joanna Reis Santos-Oliveira3, Alda Maria Da-Cruz3/+, Sonia Enosse1,2
Instituto Nacional de Saúde, Ministério da Saúde, Maputo, Moçambique 2Laboratório de Biologia Molecular,
Hospital Central de Maputo, Maputo, Moçambique 3Laboratório Interdisciplinar de Pesquisas Médicas,
Instituto Oswaldo Cruz-Fiocruz, Ministério de Saúde, Rio de Janeiro, RJ, Brasil
1
Human immunodeficiency virus (HIV)-1 infection has an important impact on malaria. Plasmodium falciparum
and HIV-1 co-infected patients (Pf/HIV) present with a high degree of anaemia, enhanced parasitaemia and decreased CD4+ T cell counts, which increase the risk of developing severe malaria. In addition, infection with either
Pf or HIV-1 alone causes extensive immune activation. Our hypothesis was that lymphocyte activation is potentiated
in Pf/HIV co-infected patients, consequently worsening their immunosuppressed state. To test this hypothesis, 22 Pf/
HIV patients, 34 malaria patients, 29 HIV/AIDS patients and 10 healthy controls without malaria or HIV/acquired
immune deficiency syndrome (AIDS) from Maputo/Mozambique were recruited for this study. As expected, anaemia
was most prevalent in the Pf/HIV group. A significant variation in parasite density was observed in the Pf/HIV
co-infected group (110-75,000 parasites/µL), although the median values were similar to those of the malaria only
patients. The CD4+ T cell counts were significantly lower in the Pf/HIV group than in the HIV/AIDS only or malaria
only patients. Lymphocyte activation was evaluated by the percentage of activation-associated molecules [CD38
expression on CD8+ and human leukocyte antigen-DR expression on CD3+ T cells]. The highest CD38 expression
was detected in the Pf/HIV co-infected patients (median = 78.2%). The malaria only (median = 50%) and HIV/AIDS
only (median = 52%) patients also exhibited elevated levels of these molecules, although the values were lower than
those of the Pf/HIV co-infected cases. Our findings suggest that enhanced T-cell activation in co-infected patients
can worsen the immune response to both diseases.
Key words: malaria/HIV-AIDS - cellular activation - Plasmodium - co-infection - CD4+ T cells - Mozambique
As leading causes of morbidity and mortality, malaria
and human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) are two of the most
important diseases in Africa (Idemyor et al. 2007). Plasmodium falciparum-associated HIV-1 (Pf/HIV) infection
has been described in many sub-Saharan Africa countries
and data from these countries account for most of the
available clinical data regarding this co-infection (Chalwe
et al. 2009, Davenport et al. 2010, Kiyingi et al. 2010, Mills
et al. 2010, Serna-Bolea et al. 2010, Naniche et al. 2011).
A relevant issue in malaria-endemic regions is the identification of HIV/AIDS patients among suspected cases of
acute malaria (Mills et al. 2010, Serna-Bolea et al. 2010).
Furthermore, HIV-1 infection is a risk factor for receiving
an incorrect diagnosis of malaria (Berg et al. 2008). Thus,
surveillance health programs should invest considerable
effort to minimise the misdiagnosis of febrile diseases.
Financial support: IOC internal founds, CAPES
HC is a student from the Post Graduate Program in Health Sciences/
Tropical Medicine, FIOCRUZ/INS-MISAU (General Cooperation
Agreement Brazil-Mozambique). JRSO is a PhD student from
PGMT and was sponsored by CNPq and FAPERJ. AMC is a
research fellow from CNPq and FAPERJ (JCNE).
+ Corresponding author: [email protected]
Received 24 March 2012
Accepted 16 August 2012
In Mozambique (Mz), malaria is endemic throughout the country and is most prevalent after the rainy
season, which lasts from December-April. Malaria and
HIV/AIDS are the most important causes of mortality
with rates of 29% and 27%, respectively (INS/INE/ICF
2010). According to the World Malaria Report (WHO
2011), approximately four million estimated cases and
3,300 deaths due to malaria were reported in Mz during
2010. Considering that 11.5% of population is estimated
to be infected with HIV-1, the overlap of these dually
endemic diseases increases for the risk of Pf and HIV-1
co-infection. Recently, a study of 136 HIV-1 infected
individuals from southwest Mz revealed that 5.1% were
also infected with Pf (Naniche et al. 2011).
There is evidence that Pf/HIV co-infection enhances the spread of both Pf malaria and HIV-1 infection and may also influence the severity of the clinical
manifestations of these diseases. Indeed, HIV infection
has been considered an important risk factor for severe
Pf malaria (Grimwade et al. 2004, Chalwe et al. 2009).
Increased Plasmodium parasitaemia is most likely related to the impairment of parasite control caused by
HIV-1-related immunosuppression (Whitworth et al.
2000, Patnaik et al. 2005). Patients with low CD4+ T
cell counts of less than 350 cells/mm 3 are more likely
to exhibit complications arising malaria (Cohen et al.
2005, Mouala et al. 2009). Moreover, HIV-1 infection is
associated with an increased prevalence of anaemia in
Pf malaria (Otieno et al. 2006, Davenport et al. 2010).
Malaria can also affect HIV-1 infection. HIV/AIDS paonline | memorias.ioc.fiocruz.br
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T-cell activation in malaria/HIV co-infection • Helena Chavale et al.
tients with malaria can exhibit a transitory reduction
in the number of CD4+ T cells, which may be partially
reversible after successful antimalarial therapy (Van
Geertruyden et al. 2006a). A causal relationship between malarial episodes and a decline in CD4+ T cell
counts in HIV-1 patients remains to be established
(Mermim et al. 2006). Acute malaria elevates the HIV
viral load (VL), which in turn can enhance the risk for
HIV transmission (Kublin et al. 2005). In addition,
Plasmodium antigens lead to strong cellular activation
(Worku et al. 1997) which may facilitate de novo HIV-1
infection and replication (Froebel et al. 2004). The preference of HIV-1 for infecting activated memory CD4+
T lymphocytes can increase cell death (Grossman et al.
2002). Consequently, it is possible that, in co-infected
patients, Pf-specific T-cell clones are depleted by HIV-1
during each malaria episode (Whitworth & Hewitt
2005, Mermim et al. 2006). Therefore, these factors
can decrease the immune response to both diseases and
contribute to HIV disease progression.
Cell activation is one of the crucial mechanisms in
the pathogenesis of both malaria (Worku et al. 1997) and
HIV-1/AIDS (Douek et al. 2009). The release of pro-inflammatory cytokines due to increased T cell activation
during acute malaria has been implicated in the clinical
complications and contributes to a disruption in the balance of effector/regulatory immune state (Ayimba et al.
2011, Davenport et al. 2012). In HIV-1/AIDS, it is well
established that the VL is directly associated with CD8+
T cell activation (Benito et al. 2004). However, the mechanism by which the association of these two pathogens
can impact the immunopathogenesis of malaria/HIVAIDS remains under discussion. Few studies addressing the compromised immunological state have been
reported for malaria/HIV-AIDS co-infection (Naniche
et al. 2011) and, to our knowledge, none has investigated
the consequences of cellular activation in the immunosuppression state. Because both Pf and HIV alone can
cause immune activation, our hypothesis was that lymphocyte activation is potentiated in co-infected patients.
As a consequence, activation-induced lymphocyte death
could aggravate the immunosuppressed state of co-in-
fected patients. For test this hypothesis, we evaluated the
severity of anaemia, Plasmodium parasite density (PD)
and impairment of the cellular immune response in malaria/ HIV-1 co-infected adults from southwest Mz.
PATIENTS, Materials and Methods
Study population - All patients were recruited from
the Polana Caniço Health Centre (PCHC) in Maputo, the
capital of Mz, between January-December 2010. PCHC
is an outpatient health care centre situated in a suburb of
Maputo and provides medical care for patients from three
neighbourhoods: Maxaquene, Polana Caniço and Hulene.
Maputo is an area of stable malaria transmission, with a
rate of ≥ one case per 1,000 inhabitants and a high percentage of individuals have a past history of malaria (73-76%).
HIV-1 prevalence in Mz is 11.5%, affecting approximately 9,000 individuals each year (INE 2009). In PCHC, the
malaria care service is responsible for the diagnosis of
malaria and the dispensation of antimalarial drugs according to the malaria management guidelines established by
Ministry of Health (MISAU)/Mz. This health centre also
attends to and counsels HIV/AIDS patients.
A cross-sectional study was performed in which Pf/
HIV-1 co-infected patients were recruited from patients
presenting with clinical malaria. A total of 99 recruited
participants were placed into the following four groups:
22 Pf/HIV, 29 patients infected with HIV-1 only (HIV-1/
AIDS), 38 infected patients with Pf only (malaria) and a
group of 10 subjects who were negative for both malaria
and HIV-1 [healthy controls (HC)]. The demographic
characteristics of the study subjects are shown in Table.
Their ages ranged from 20-75 years (mean age - 38 ± 12.2;
median - 37 years) and the majority of the participants
were female (59.2%). Laboratory exams were performed
to diagnose anaemia, malaria and HIV/AIDS according
to the MISAU/Mz guidelines. The levels of haemoglobin
(Hg) and white blood cells (WBC) were determined by a
complete haemogram exam. The results were expressed
as g/dL for Hg and 103/µL for WBC.
The patients were screened for malaria with a qualitative rapid diagnostic test (RDT) (SD Malaria Antigen
Pf BIOLINE kit, Standard Diagnosis Inc, Giheung-ku,
TABLE
Demographic characteristics of study population
Distribution per gender
Cases/controls
Pf/HIV co-infected
Malaria
HIV/AIDS
Healthy controlsb
Cases
(n)
Age (years)
mean ± SDa (median)
Male (40.7%)
(%)
Female (59.2%)
(%)
22
38
29
10
38.2 ± 15 (34)
39.4 ± 13 (39)
37 ± 10.8 (36)
34.5 ±7 (34)
36.4
31.6
51.7
50
63.6
68.4
48.3
50
a: no statistical difference among the four groups; b: subjects without malaria or human immunodeficiency virus (HIV)-1/acquired
immune deficiency syndrome (AIDS); Pf/HIV: Plasmodium falciparum and HIV-1 co-infected patients; SD: standard deviation.
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(8), December 2012
Republic of South Korea) to detect the histidine rich
protein II of Pf in blood samples. After their consent,
patients with a RDT positive test were screened for HIV
according to the Mozambican National protocol. This
protocol consists of a sequential algorithm of two immunochromatographic rapid tests. All individuals were
first screened with the Determine HIV-1/2 test (Abbott
Laboratories Co, Ltd, Tokyo Japan). All specimens that
were reactive by this screening assay were further tested
using the HIV test, Uni-goldTM Recombigen® (Trinity
Biotech PLC, Bray Ireland). Patients over 18 years of
age, who were infected with HIV-1, who did not receive
antiretroviral (ARV) therapy and who had a positive
RDT for malaria were recruited for the co-infected study
group. Pregnancy tests were used to identify pregnant
women that were excluded from the study. Patients who
were positive for only malaria or HIV-1 infection were
enrolled into either the malaria or HIV/AIDS groups, respectively. Volunteers who were negative for both tests
comprised the HC group. HIV/AIDS patients were referred to the counselling program.
Determination of Plasmodium PD - The Plasmodium
species identification and PD were determined in RDT
positive patients. To determine the malarial parasite density, the finger was pricked and a thick blood smear was
made. The blood smears were stained with Giemsa stain
and were examined under oil-immersion microscope by
trained laboratory technicians until had greater than 500
WBC or 500 parasites. The Pf PD was calculated after
the actual leukocyte count was taken into consideration and was determined by the following formula: PD
= (number of parasites/number WBC on each slide) x
WBC per mL from each patient x 103. The results were
expressed as the number of parasites/µL.
Determination of absolute T cells counts and lymphocyte activation levels - To quantify the number of
CD4+ T and CD8+ T lymphocytes in the blood, BD Tritest® monoclonal antibodies specific for CD4, CD8 and
CD3 conjugated to FITC, PE and PerCP, respectively
and the BDTrue Count® reagent kit were used according to the manufacturer’s instructions (BD Biosciences,
Franklin Lakes, NJ, USA). In parallel, the blood cells
were also stained with anti-CD8 FITC/anti-CD38 PE
and anti-CD3 FITC/anti-human leukocyte antigen
(HLA)-DR PE monoclonal antibodies (BD SimultestTM,
BD Biosciences, San Jose, CA, USA).
The samples were acquired using a FACSCalibur ®
flow cytometer (BD, USA) and analysed by Multiset®
software for CD4 counting, whereas phenotypic analysis was performed with by CellQuestTM software. Both
software programs were obtained from BD Biosciences.
The experiments were performed at the centre of reference for AIDS diagnosis at the MISAU/Mz [Laboratory
of Molecular Biology and Hematology at Maputo Central Hospital, Laboratory of Parasitology and Immunology National Institutes of Health (INS)] and in Brazil
[Laboratory of Molecular Immunology and AIDS, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (IOC/
FIOCRUZ), Ministry of Health].
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Although the validated marker of CD8+ T cell activation is the co-expression of CD38 and HLA-DR, activated cells were identified by detecting CD38 on CD8+
T cells and HLA-DR on CD3+ T cells. These molecules
were used as surrogates for the validated marker (Douek
et al. 2009). HLA-DR positivity was determined on the
CD3+ T cell population and CD38 positivity was determined on the CD8+ T cell population, inside the gate previously established for the CD3+ T cell population. The
results were expressed as the percentage of HLA-DR on
CD3+ and CD38 on CD8high T cells.
Statistical analysis - The values were expressed as
the median and interquartile range (IQR). The MannWhitney U test and the non-parametric Spearman’s test
were used for the comparison and correlation analysis
of the data, respectively (GraphPad Prism, version 4.0,
San Diego, CA, USA). The differences were considered
significant when p < 0.05.
Ethics - The study was approved by the Mozambican
Ethical Committee (National Bioethics Committee for
Health), under protocol 194/CNBS and written informed
consent was obtained from all participants.
RESULTS
Impact of malaria-associated HIV-1/AIDS on the severity of anaemia - Hg levels were used to evaluate the
degree of anaemia (Fig. 1). Co-infected patients had lower
Hg levels (median = 10.1 g/dL, IQR - 8.4-11.2 g/dL) than
did patients with Pf only (median = 12g/dL, IQR - 10.213.3 g/dL, p < 0.001) or HIV-1/AIDS only (median = 11.4
g/dL, IQR - 11.2-12.6 g/dL, p < 0.05) or the HC group
(median = 12.8, IQR - 11.5-14 g/dL n = 10, p < 0.01) (Fig.
1). No significant differences were observed between the
malaria and HIV-1/AIDS only patients or between the
HC and the malaria or HIV-1/AIDS only patients.
Fig. 1: anaemia severity estimated by haemoglobin concentration in
Plasmodium falciparum and human immunodeficiency virus (HIV)-1
co-infected (Pf/HIV)/acquired immune deficiency syndrome (AIDS)
patients. Pf/HIV co-infected patients (black and white circles), Pf
malaria only patients (white circles) and HIV-1/AIDS only patients
(black circles) and healthy controls (HC) without malaria or HIV-1/
AIDS (solid triangles). Each point represents one subject. The horizontal bars express median.
988
T-cell activation in malaria/HIV co-infection • Helena Chavale et al.
Fig. 2: determination of Plasmodium falciparum (Pf ) parasite density
(PD) in human immunodeficiency virus (HIV)-1 co-infected patients.
The blood smears were stained with Giemsa stain and the parasites
were counted until the observed fields had reached greater than 500
white blood cells or 500 parasites. The PD was calculated according to the formula shown in Patients, Materials and Methods section,
considering individual actual leukocytes counts per mL. Black and
white circles: Pf/HIV co-infected patients; white circles: Pf malaria
only patients. Each point represents one subject. The horizontal bars
express median. There was no statistic difference between groups.
Fig. 3: evaluation of immunosuppressed state in malaria associated
human immunodeficiency virus (HIV)-1/acquired immune deficiency syndrome (AIDS) patients by the absolute number of CD4+ T
lymphocytes. Black and white circles: Plasmodium falciparum (Pf )/
HIV co-infected patients; black circles: HIV-1/AIDS only patients;
solid triangles: healthy controls (HC) without malaria or HIV-1/
AIDS; white circles: Pf malaria only patients. Each point represents
one subject. The horizontal bars express median.
PD in Pf/HIV - Fifty-nine patients were positive for
malaria by RDT; however the PD quantification was
possible for only 33 of these patients (Fig. 2). There was
no significant difference in the PD of the co-infected patients (median = 8,326 parasites/µL, IQR - 1,087-20,357
parasites/µL, n = 14) compared to the malaria only patients (median = 3,308 parasites/µL, IQR - 1,013-8,050
parasites/µL, n = 19). Although, high levels of parasitaemia were observed in both groups, a wider variation
in the parasite load was observed in the co-infected patients (110-75,000 parasites/µL).
Malaria-associated HIV-1 co-infected patients
present with low CD4+ T lymphocytes levels - To determine whether the malaria association may influence
the immunosuppression caused by HIV-1 infection, the
blood levels of CD4+ T lymphocytes were quantified
(Fig. 3). Significantly lower CD4+ T values were observed in the Pf/HIV co-infected patients (median =
265 cells/mm 3, IQR - 195-545 cells/mm 3, n = 22) than
in the HIV-1/AIDS patients (median = 406 cells/mm 3,
IQR-274-543 cells/mm 3, n = 29, p < 0.05). These levels
were also lower than those observed in the malaria patients (median = 597 cells/mm3, IQR - 318-868 cells/
mm3, n = 38, p < 0.01) or the HC group (median = 1,084
cells/mm3, IQR - 1,007-1,224 cells/mm 3, n = 10, p <
0.001). In addition, HIV-1/AIDS patients also exhibited reduced CD4+ T cell levels compared to malaria
patients (p < 0.05) or HC (p < 0.001). A positive correlation between CD4+ T cell counts and Hg levels were
observed when all groups were evaluated together (r =
0.40, p < 0.001) (data not shown).
The association of malaria plus HIV-1 enhances the
T cell activation state - To determine whether Pf infection may contribute to augmentation T cell activation in
Pf/HIV co-infected patients, the percentage of activated
CD3+ and CD8+ T cells was measured by flow cytometry
using the expression of the activation-associated membrane molecules HLA-DR and CD38, respectively.
Fig. 4: T lymphocyte activation state in Plasmodium falciparum and human immunodeficiency virus (HIV)-1 co-infected (Pf/HIV)/acquired
immune deficiency syndrome (AIDS) patients. Black and white circles: Pf/HIV co-infected patients; black circles: HIV-1/AIDS only patients;
solid triangles: healthy controls (HC) without malaria or HIV-1/AIDS; white circles: Pf malaria only patients. The flow cytometry analysis of
the percentage of human leukocyte antigen-DR on CD3+ T (A) and CD38 on CD8+ T (B) cells were determined in lymphocyte gates as described
in Patients, Materials and Methods section. Each point represents one subject. The horizontal bars express median.
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(8), December 2012
Pf/HIV co-infected patients presented with the highest levels of lymphocyte activation compared to the other
groups (Fig. 4A). The levels of HLA-DR+ on the CD3+ T
cells from the Pf/HIV patients were significantly higher
(median = 67.5%, IQR - 39.3-77.8%, n = 17) than on T
cells from the malaria only patients (median = 32.1%,
IQR - 21.2-45%, n = 29, p < 0.001) or the HIV-AIDS only
patients (median = 48.8%, IQR - 28.8-57%, n = 13, p <
0.05). Notably, the malaria patients also exhibited high
levels of HLA-DR+ on CD3+ T cells, although the levels were lower than those observed on cells from HIV-1/
AIDS patients (p < 0.05).
With regard to the percentage of CD38+ on CD8+ T
lymphocytes (Fig. 4B), the state of cellular activation
was also more elevated in the Pf/HIV co-infected group
(median = 78.2%, IQR - 43.5-85%, n = 17) than in the
malaria only patients (median = 50%, IQR - 37.3-61.5%,
n = 29, p < 0.01). However, no significant difference was
observed with regard to the HIV-1/AIDS only group
(median = 52%, IQR - 49.8-73.1%, n = 13). Elevated
levels of activation were observed in the malaria and
HIV-1/AIDS only patients and there were no difference
between these groups. As expected, the lowest levels
of cellular activation were observed in the HC group
(HLA-DR+, median = 3.4%, IQR - 2.6-7% and CD38+,
median = 28.9%, IQR - 25-35.3%).
To determine if the parasitaemia is involved in the
cellular activation of Pf/HIV co-infected patients, a
correlation analysis was performed. For this analysis
17 Pf/HIV co-infected patients and 29 malaria only patients were studied. No significant correlation between
PD and the percentage of HLA-DR+ and CD38+ on the
T lymphocytes was observed for either group. By contrast, a negative correlation was observed between the
CD4+ T cell counts and HLA-DR on the CD3+ T lymphocytes when malaria, HIV-AIDS, Pf/HIV co-infected patients and the HC group were considered together
(r = -0.70, p < 0.01) (Fig. 5).
Fig. 5: correlation between CD4+ T cell counts and human leukocyte
antigen (HLA)-DR+ on CD3+ T lymphocytes in Plasmodium falciparum and human immunodeficiency virus (HIV)-1 co-infected (Pf/
HIV)/acquired immune deficiency syndrome (AIDS) patients. Black
and white circles: Pf/HIV co-infected patients; black circles: HIV-1/
AIDS only patients; solid triangles: healthy controls (HC) without
malaria or HIV-1/AIDS; white circles: Pf malaria only patients.
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DiscussioN
Most of the available data regarding malaria-associated HIV-AIDS is related to the consequences of HIV
infection on the degree of anaemia, Plasmodium PD and
CD4+ T lymphocyte counts in Pf malaria patients, particularly in sub-Saharan Africa countries (Whitworth et
al. 2000, Cohen et al. 2005, Patnaik et al. 2005, Mouala
et al. 2009, Davenport et al. 2010). However, the impact
of both infections on the state of immune cellular activation is largely unknown. This study investigated whether
co-infection had a synergistic effect on the immune activation induced by Plasmodium or HIV-1 alone. Our
results suggest that Pf malaria enhances T lymphocyte
activation in individuals concomitantly infected with
HIV-1. This phenomenon can affect immune effector
function and may constitute an additional mechanism
that contributes to the pathogenesis of malaria-associated HIV-1/AIDS disease.
Anaemia is a major symptom of malaria and can be
caused by several mechanisms, including the destruction of infected erythrocytes, antibody-mediated lysis,
hypersplenism, impaired erythropoiesis and cytokines
(Idemyor et al. 2007, Haldar & Mohandas 2009, Quintero et al. 2011). In a previous study, 10% of adults with
severe malaria had Hg levels of 7 g/dL or less, which
is much lower than expected for a healthy individual
(WHO 2000). Moreover, HIV-1 infection itself can also
cause anaemia (Belperio & Rhew 2004) and constitutes
an important risk factor for severe malaria in adults
(Grimwade et al. 2004, Chalwe et al. 2009). In our study,
anaemia was present at a higher degree in Pf/HIV coinfected patients, since they had lower Hg levels than
the malaria only patients. This difference does not seem
to be related to gender as the percentage of females was
higher than males in both groups. In addition, the Hg
concentration and PD are not correlated in the Pf/HIV
co-infected patients or the malaria only patients. These
results are consistent with other studies of malaria transmission in sub-Saharan Africa areas, such as Cameroon
(Nkuo Akenji et al. 2002) and Kenya (Davenport et al.
2010). However, conflicting results regarding anaemia
have also been observed in Kenya (Mc Elroy et al. 2000)
and Tanzania (Ekval et al. 2001). As has been shown for
other conditions, such as malnutrition, ARV drugs use
or infection with intestinal parasites, co-infection with
HIV and malaria can worsen the anaemic condition, particularly in low income regions.
Immunosuppression caused by HIV infection appears to impair the ability to control the parasitaemia,
resulting in an enhancement of the Plasmodium PD (Van
Geertruyden et al. 2006b). Despite reports that HIV individuals are more likely to be parasitaemic (Whitworth
et al. 2000, Patnaik et al. 2005), there was no significant difference in the parasite load between the Pf/HIV
co-infected patients and the malaria only patients is this
study. It is important to note that, in our study, the parasitaemia intensity calculation was based on the individual leukocyte count per mL instead of a fixed number of
leukocytes per mL. Notably, a wide range of PD levels
was detected in the Pf/HIV co-infected group, varying
from 110-75,000 parasites/µL.
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T-cell activation in malaria/HIV co-infection • Helena Chavale et al.
Depletion of CD4+ T cells is a common consequence
of HIV-1 infection (Grossman et al. 2002). However, acute
malaria only also reduces the number CD4+ T lymphocytes (Chougnet et al. 1992, Kassa et al. 2006). Among
the patients evaluated in this study, the lowest CD4+ T
values were observed in the co-infected patients. The
number of CD4+ T cells in the malaria or HIV-1/AIDS
only patients was also lower than in the HC group. These
results suggest a heightened degree of immunosuppression in malaria-associated HIV-1/AIDS. Interestingly,
this reduction in CD4+ T cells is transient and the levels
of these cells increase after successful antimalarial therapy, although the number of CD4+ T cells does not reach
the previous counts (Kassa et al. 2006, Van Geertruyden
et al. 2006a. Therefore, each episode of acute malaria
causes an excess decrease in the CD4+ T cell numbers
(Mermim et al. 2006), which aggravates the immunosuppressed state. These facts highlight the importance
of assessing any malaria-induced disturbances in the
laboratory prior to deciding how to manage HIV positive
individuals. This approach can avoid the misdiagnosis of
ARV failure or even the premature initiation of therapy
(Brentlinger et al. 2007). Unfortunately, access to ARV
is not yet universal, either in Mz (INSIDA 2009) or in
other African countries, whereas antimalarials drugs are
currently accessible for the population.
CD4+ T cell counts have been implicated in Plasmodium control (Brentlinger et al. 2007, Idemyor 2007),
because these cells are negatively correlated with PD
(Withworth et al. 2000, Patnaik et al. 2005). However,
we did not observe any association between these parameters, perhaps because of the study sample size.
Moreover, because the adult population evaluated in this
study is from a stable transmission area, these adults are
considered immune and therefore, the level of parasitaemia should be lower than in children from the same area,
potentially explaining the lack of correlation between
the PD and immunosuppression.
Chronic immune activation is the strongest predictor
of the progression rate to AIDS in HIV-1 infected individuals (Douek et al. 2009). Acute malaria infection may
also be associated with an enhanced cellular activation
state. Co-expression of CD38/HLA-DR on T cell subsets
has previously been studied in both HIV-1/AIDS or malaria alone (Hunt et al. 2003, Eggena et al. 2006, Naniche
et al. 2011) and immune activation was considered a predictor of treatment failure in malaria patients (Eggena et
al. 2006). Malaria infection can present with a pro-inflammatory response and cytokine imbalance (Ayimba
et al. 2011, Davenport et al. 2012). In this study, Pf/HIV
co-infected patients exhibited the highest percentages of
HLA-DR and CD38 on the CD3+ and CD8+ T lymphocytes, respectively. Indeed, malaria patients had higher
percentages of CD38 on CD8+ and HLA-DR on CD3+ T
cells than did the healthy subjects, confirming previous
data that malaria itself contributes to activation (Worku
et al. 1997). Although dual-positive CD38/HLA-DR on
CD8+ T cells is a more accurate measure of the activation
state, when both molecules were analysed separately,
they displayed the same profile. Similar to that observed
for the CD4+ T counts, the viral or parasite antigens ap-
pear to act together to boost cell activation, either directly or by inducing the release of soluble factors. HIV-1
co-infection with other diseases, such as leishmaniasis
(Santos-Oliveira et al. 2010), tuberculosis (Rodrigues et
al. 2003) or leprosy (Giacoia-Gripp et al. 2011), has previously been demonstrated to increase cellular activation. A previous study conducted in Mz did not identify
a significant correlation between the “malaria infection”
parameter and the levels of HLA-DR/CD38 on activated
CD4+ and CD8+ T cells (Naniche et al. 2011). However,
the authors of this study did not compare Pf/HIV coinfected patients to malaria or HIV/AIDS only patients.
We cannot exclude the possibility that dual-infected PfHIV patients could be in more advanced stages of HIV/
AIDS and/or have more opportunistic infections, which
could contribute to decreased CD4+ T cell counts and
increased T cell activation.
Viral replication is a well-known factor that contributes to lymphocyte activation and is considered a
predictor parameter for HIV VL (Benito et al. 2004).
Nevertheless, one limitation of our study is that we did
not determine the HIV-1 VL, because this assessment is
not routinely available for African public health systems
(INSIDA 2009). This study did not find a causal relationship between Pf PD and T cell activation. However,
malarial antigens cannot be ruled out as an important
factor for immune activation. Therefore, the role of each
infection in this exacerbated activation state and the additional implications for the pathogenesis and management of malaria/HIV-1 co-infection must be clarified. In
addition, we did not follow-up the Pf/HIV co-infected
patients during malaria, their convalescence to evaluate whether the T-cell activation state decreased and
achieved the activation levels observed in the malaria or
HIV-1/AIDS only patients.
Our results demonstrate that the anaemia, Pf parasitaemia and CD4+ T counts profiles of malaria-HIV-1
infected patients from southwest Mz are similar to those
observed in other sub-Saharan Africa studies (Patnaik et
al. 2005, Van Geertruyden et al. 2006a, Davenport et al.
2010). Complementary to these studies, our findings suggest that malaria/HIV-1-AIDS association may contribute to enhanced T cell activation in co-infected patients.
This phenomenon is of utmost importance because these
cells can (i) augment the release of proinflammatory cytokines causing systemic damage and (ii) be more susceptible to die by activation-induced cell death (Kemp
et al. 2002). Finally, potential regional differences are
expected to occur depending on the malaria endemicity. Additional studies in sub-Saharan Africa countries,
including prospective follow-up are of extreme importance to better understand the relationship between malaria and HIV-1 infection and the consequences of these
infections on the immunopathogenesis of malaria/HIV-1
co-infection.
ACKNOWLEDGEMENTS
To Dr Simone Ladeia, for critical reviewing the paper, to
the health workers from Polana Caniço Health Centre outpatient unit, for helping recruitment of patients, to Ms. Elisa M
Mahanjane, for her helpful secretarial assistance, to the Labora-
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(8), December 2012
tory of Parasitology and Immunology (INS/MISAU) and to Dr
Mariza Morgado (Laboratory of Molecular Immunology and
AIDS-IOC/Fiocruz), for allowing the use of the equipment and
their technical academic support, and to Dr Ilesh Jani and Dr
Wilson Savino (coordinators of PGCS, INS/MISAU, Fiocruz),
for their support during the development of this study.
References
Ayimba E, Hegewald J, Ségbéna AY, Gantin RG, Lechner CJ, Agosssou A, Banla M, Soboslay PT 2011. Proinflammatory and regulatory cytokines and chemokines in infants with uncomplicated
and severe Plasmodium falciparum malaria. Clin Exp Immunol
166: 218-226.
Belperio PS, Rhew DC 2004. Prevalence and outcomes of anemia in
individuals with human immunodeficiency virus: a systematic
review of the literature. Am J Med 116 (Suppl 7A): 27S-43S.
Benito JM, Lopez M, Lozano S, Martinez P, Gonzalez-Lahoz J, Soriano V 2004. CD38 expression on CD8+ T lymphocytes as a
marker of residual virus replication in chronically HIV-infected
patients receiving antiretroviral therapy. AIDS Res Hum Retroviruses 20: 227-233.
Berg A, Patel S, Langeland N, Blomberg B 2008. Falciparum malaria
and HIV-1 in hospitalized adults in Maputo, Mozambique: does
HIV-infection obscure the malaria diagnosis? Malar J 7: 252.
Brentlinger PE, Behrens CB, Kublin JG 2007. Challenges in the prevention, diagnosis and treatment of malaria in human immunodeficiency virus infected adults in sub-Saharan Africa. Arch Intern
Med 167: 1827-1836.
Chalwe V, Van Geertruyden JP, Mukwamataba D, Menten J, Kamalamba J, Mulenga M, D’Alessandro U 2009. Increased risk for
severe malaria in HIV-1-infected adults, Zambia. Emerg Infect
Dis 15: 749.
Chougnet C, Tallet S, Ringwald P, Deloron P 1992. Kinetics of lymphocyte subsets from peripheral blood during a Plasmodium falciparum malaria attack. Clin Exp Immunol 90: 405-408.
Cohen C, Karstaedt A, Frean J, Thomas J, Govender N, Prentice E,
Dini L, Galpin J, Crewe-Brown H 2005. Increased prevalence of
severe malaria in HIV-infected adults in South Africa. Clin Infect
Dis 41: 1631-1637.
Davenport GC, Hittner JB, Were T, Ong’echa JM, Perkins DJ 2012.
Relationship between inflammatory mediator patterns and anemia in HIV-1 positive and exposed children with Plasmodium
falciparum malaria. Am J Hematol 87: 652-658.
Davenport GC, Ouma C, Hittner JB, Were T, Ouma Y, Ong’echa JM,
Perkins DJ 2010. Hematological predictors of increased severe
anemia in Kenyan children coinfected with Plasmodium falciparum and HIV-1. Am J Hematol 85: 227-233.
Douek D, Roederer M, Koup R 2009. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med 60: 471-484.
Eggena MP, Hopkins H, Barugahare B, Okello M, Ssali F, Mugyenyi
P, Rosenthal PJ, Cao H, Dorsey G 2006. CD4 T cell activation as a
predictor for treatment failure in Ugandans with Plasmodium falciparum malaria. Am J Trop Med Hyg 74: 41-43.
Ekvall H, Premji Z, Bennett S, Bjorkman A 2001. Hemoglobin concentration in children in a malaria holoendemic area is determined by cumulated Plasmodium falciparum parasite densities.
Am J Trop Med Hyg 64: 58-66.
Froebel K, Howard W, Schafer JR, Howie F, Whitworth J, Kaleebu P,
Brown AL, Riley E 2004. Activation by malaria antigens renders
mononuclear cells susceptible to HIV infection and re-activates
replication of endogenous HIV in cells from HIV-infected adults.
Parasite Immunol 26: 213-217.
991
Giacoia-Gripp CB, Sales AM, Nery JA, Santos-Oliveira JR, de Oliveira AL, Sarno EN, Morgado MG 2011. Evaluation of cellular
phenotypes implicated in immunopathogenesis and monitoring
immune reconstitution inflammatory syndrome in HIV/leprosy
cases. PLoS ONE 6: e28735.
Grimwade K, French N, Mbatha DD, Zungu DD, Dedicoat M, Gilks
CF 2004. HIV infection as a cofactor for severe falciparum malaria in adults living in a region of unstable malaria transmission
in South Africa. AIDS 18: 547-554.
Grossman Z, Meier-Schellersheim M, Sousa AE, Victorino RM, Paul
WE 2002. CD4+ T-cell depletion in HIV infection: are we closer
to understanding the cause? Nat Med 8: 319-323.
Haldar K, Mohandas N 2009. Malaria, erythrocytic infection and
anemia. Hematology Am Soc Hematol Educ Program 87-93.
Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, Lampiris H,
Deeks SG 2003. T cell activation is associated with lower CD4+
T cell gains in human immunodeficiency virus-infected patients
with sustained viral suppression during antiretroviral therapy.
J Infect Dis 187: 1534-1543.
Idemyor V 2007. Human immunodeficiency virus (HIV) and malaria
interaction in sub-Saharan Africa: the collision of two Titans.
HIV Clin Trials 8: 246-253.
INE - Instituto Nacional de Estatística 2009. Mortalidade em Moçambique: inquérito nacional sobre as causas de mortalidade,
2007/2008 - relatório preliminar, INE, Moçambique, 100 pp.
INSIDA - Instituto Nacional de Saúde 2009. Inquérito nacional de
prevalência, riscos comportamentais e informação sobre o HIV
e SIDA em Moçambique, INSIDA, Maryla, 310 pp.
Kassa D, Petros B, Mesele T, Hailu E, Wolday D 2006. Characterization of peripheral blood lymphocyte subsets in patients with acute
Plasmodium falciparum and P. vivax malaria infections at Wonji
Sugar Estate, Ethiopia. Clin Vaccine Immunol 13: 376-379.
Kemp K, Akanmori BD, Adabayeri V, Goka BQ, Kurtzhals JA, Behr
C, Hviid L 2002. Cytokine production and apoptosis among T
cells from patients under treatment for Plasmodium falciparum
malaria. Clin Exp Immunol 127: 151-157.
Kiyingi HS, Egwang TG, Nannyonga M 2010. Prolonged elevation
of viral loads in HIV-1-infected children in a region of intense
malaria transmission in Northern Uganda: a prospective cohort
study. Pan Afr Med J 7: 11.
Kublin JG, Patnaik P, Jere CS, Miller WC, Hoffman IF, Chimbiya
N, Pendame R, Taylor TE, Molyneux ME 2005. Effect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in
the blood of adults in rural Malawi: a prospective cohort study.
Lancet 365: 233-240.
Mc Elroy PD, ter Kuile FO, Lal AA, Bloland PB, Hawley WA, Oloo
AJ, Monto AS, Meshnick SR, Nahlen BL 2000. Effect of Plasmodium falciparum parasitemia density on hemoglobin concentrations among full-term, normal birth weight children in western
Kenya. IV. The Asembo Bay Cohort Project. Am J Trop Med Hyg
62: 504-512.
Mermin J, Lule JR, Ekwaru JP 2006. Association between malaria
and CD4 cell count decline among persons with HIV. J Acquir
Immune Defic Syndr 41: 129-130.
Mills LA, Kagaayi J, Nakigozi G, Galiwango RM, Ouma J, Shott JP,
Ssempijja V, Gray RH, Wawer MJ, Serwadda D, Quinn TC, Reynolds SJ 2010. Utility of a point-of-care malaria rapid diagnostic test
for excluding malaria as the cause of fever among HIV-positive
adults in rural Rakai, Uganda. Am J Trop Med Hyg 82: 145-147.
992
T-cell activation in malaria/HIV co-infection • Helena Chavale et al.
Mouala C, Guiguet M, Houzé S, Damond F, Pialoux G, Viget N,
Costagliola D, Le Bras J, Matheron S, FHDH-ANRS CO4 Clinical Epidemiology Group 2009. Impact of HIV infection on severity of imported malaria is restricted to patients with CD4 cell
counts < 350 cells/microl. AIDS 23: 1997-2004.
Naniche D, Letang E, Nhampossa T, David C, Menendez C, Alonso
P 2011. Alterations in T cell subsets in human immunodeficiency
virus-infected adults with co-infections in southern Mozambique. Am J Trop Med Hyg 85: 776-781.
Nkuo Akenji TK, Ajame EA, Achidi EA 2002. An investigation of
symptomatic malaria parasitaemia and anaemia in nursery and
primary school children in Buea District Cameroon. Cent Afr J
Med 48: 1-4.
Otieno RO, Ouma C, Ong’echa JM, Keller CC, Were T, Waindi EN,
Michaels MG, Day RD, Vulule JM, Perkins DJ 2006. Increased
severe anemia in HIV-1-exposed and HIV-1-positive infants and
children during acute malaria. AIDS 20: 275-280.
Patnaik P, Jere CS, Miller WC, Hoffman IF, Wirima J, Pendame R,
Meshnick SR, Taylor TE, Molyneux ME, Kublin JG 2005. Effects of HIV-1 serostatus, HIV-1 RNA concentration and CD4
cell count on the incidence of malaria infection in a cohort of
adults in rural Malawi. J Infect Dis 192: 984-991.
Quintero JP, Siqueira AM, Tobón A, Blair S, Moreno A, ArévaloHerrera M, Lacerda MVG, Valencia SH 2011. Malaria-related
anaemia: a Latin American perspective. Mem Inst Oswaldo Cruz
106 (Suppl. I): 91-104.
Rodrigues DS, Cunha RM, Kallas EG, Salomão R 2003. Distribution
of naive/effector CD4+ T lymphocytes and expression of CD38 on
CD8+ T lymphocytes in AIDS patients with tuberculosis. Braz J
Infect Dis 7: 161-165.
Santos-Oliveira JR, Giacoia-Gripp CB, Alexandrino de Oliveira P,
Amato VS, Lindoso JÂ, Goto H, Oliveira-Neto MP, Mattos MS,
Grinsztejn B, Morgado MG, Da-Cruz AM 2010. High levels of
T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load. BMC Infect Dis 10: 358.
Serna-Bolea C, Muñoz J, Almeida JM, Nhacolo A, Letang E, Nhampossa T, Ferreira E, Alonso P, Naniche D 2010. High prevalence
of symptomatic acute HIV infection in an outpatient ward in
southern Mozambique: identification and follow-up. AIDS 24:
603-608.
Van Geertruyden JP, Mulenga M, Kasongo W, Polman K, Colebunders R, Kestens L, D’Alessandro U 2006a. CD4 T-cell count
and HIV-1 infection in adults with uncomplicated malaria. J Acquir Immune Defic Syndr 43: 363-367.
Van Geertruyden JP, Mulenga M, Mwananyanda L, Chalwe V, Moerman F, Chilengi R, Kasongo W, Van Overmeir C, Dujardin JC,
Colebunders R, Kestens L, D’Alessandro U 2006b. HIV-1 immune suppression and antimalarial treatment outcome in Zambian adults with uncomplicated malaria. J Infect Dis 194: 917-925.
Whitworth J, Morgan D, Quigley M, Smith A, Mayanja B, Eotu H,
Omoding N, Okongo M, Malamba S, Ojwiya A 2000. Effect of
HIV-1 and increasing immunosuppression on malaria parasitaemia and clinical episodes in adults in rural Uganda: a cohort
study. Lancet 356: 1051-1056.
Whitworth JA, Hewitt KA 2005. Effect of malaria on HIV-1 progression and transmission. Lancet 365: 196-197.
WHO - World Health Organization 2000. Communicable diseases
cluster 2000. Trans R Soc Trop Med Hyg 94 (Suppl 1): S1-S90.
WHO - World Health Organization 2011. World malaria Report 2011. Available from: whqlibdoc.who.int/publications/2010/9789241564106_
eng.pdf>.
Worku S, Björkman A, Troye-Blomberg M, Jemaneh L, Färnert A,
Christensson B 1997. Lymphocyte activation and subset redistribution in the peripheral blood in acute malaria illness: distinct
gammadelta+ T cell patterns in Plasmodium falciparum and P.
vivax infections. Clin Exp Immunol 108: 34-41.